Gene therapy involves using nucleic acid as a pharmaceutical agent to treat disease by introducing a target gene into cells and may become an effective treatment in cases where current treatments are insufficient. Recently, the RNA interference (RNAi) field has emerged as a new therapy approach. RNAi is a naturally occurring process of sequence-specific post transcriptional gene silencing, by which gene expression is inhibited RNAi offers tremendous therapeutic promise to silence genes that exhibit aberrant behavior and therefore cause disease. While the number of possible targets for this type of treatment is increasing, clinical success is still rare, due in part to imperfect delivery systems.
A major challenge in gene therapy is finding efficient ways to introduce the desired genes into target cells in a stable manner. Understanding the transfection process is essential in order to improve the efficiency of gene therapy. The transfection process includes introduction of a complex of carrier and nucleic acid which binds to the cell membrane, endocytotic cellular uptake of the complex by the plasma membrane, escape of the nucleic acid from the endosome (endosomal release) into the cell cytoplasm, and complex unpacking. In the case of DNA transfection, the process also includes a crucial step of transport to the nucleus and entrance of the DNA into the nucleus. In the case of RNA transfection, the rate-limiting steps appear to be the endosomal release and unpacking.
Non-viral gene delivery systems have become increasingly desirable in both, basic research and clinical settings as they eliminate some of the problems associated with viral vectors. Presently, non-viral carriers used for gene transfer consist mostly of liposomal formulations and synthetic cationic polymers. Non-viral gene delivery systems based on natural polysaccharides may be advantageous over the current available synthetic ones, due to several characteristics, such as biodegradability, biocompatibility, low immunogenicity and minimal cytotoxicity. However, in contrast with the abundance of structurally different synthetic polymers, there is only a small number of polycations of a natural origin available.
Several natural or modified cationic polysaccharides have previously been tested as carriers for DNA transfection. Azzam et al., 2004 tested dextran polysaccharide modified by grafting with mixtures of spermine and other natural/synthetic oligoamines of two to four amine groups, Lee et al., 2001 found that water-soluble low molecular weight chitosan is an efficient carrier for DNA delivery, and Mansouri et al., 2004 summarizes the use of chitosan as a carrier for DNA transfection.
In an earlier work (Sieradzki et al., 2008) the present inventors modified polysaccharides into cationic polysaccharides by the process of quaternization, which is the introduction of quaternary ammonium groups to the polysaccharide. The cationic polysaccharide has permanent or induced cationic charge and was shown to interact electrostatically with the negatively charged DNA to form complexes, thus condensing it effectively for delivery into cells.